title: Study of neurotrophin-3 signaling in primary cultured neurons using multiplex stable isotope labeling with amino acids in cell culture.
abstract: Conventional stable isotope labeling with amino acids in cell culture (SILAC) requires extensive metabolic labeling of proteins and therefore is difficult to apply to cells that do not divide or are unstable in SILAC culture. Using two different sets of heavy amino acids for labeling allows for straightforward SILAC quantitation using partially labeled cells because the two cell populations are always equally labeled. Here we report the application of this labeling strategy to primary cultured neurons. We demonstrated that protein quantitation was not compromised by incomplete labeling of the neuronal proteins. We used this method to study neurotrophin-3 (NT-3) signaling in primary cultured neurons. Surprisingly our results indicate TrkB signaling is a major component of the signaling network induced by NT-3 in cortical neurons. In addition, involvement of proteins such as VAMP2, Scamp1, and Scamp3 suggests that NT-3 may lead to enhanced exocytosis of synaptic vesicles.
authors.affiliation: Kimmel Center for Biology and Medicine at the Skirball Institute, New York, New York 10016, United States.
authors.forename: Guoan
authors.initials: G
authors.lastname: Zhang
authors.forename: Katrin
authors.initials: K
authors.lastname: Deinhardt
authors.forename: Moses V
authors.initials: MV
authors.lastname: Chao
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Neubert
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002853:Chromatography, Liquid; D019295:Computational Biology; D004591:Electrophoresis, Polyacrylamide Gel; D005333:Fetus; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D007553:Isotope Labeling / Q000379:methods*; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D009474:Neurons / Q000378:metabolism*; D020933:Neurotrophin 3 / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D020813:Receptor, trkB / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*; D053719:Tandem Mass Spectrometry; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism
chemical_list: D000596:Amino Acids; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D020933:Neurotrophin 3; C083429:Scamp1 protein, rat; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2; D020813:Receptor, trkB